The relative contribution of mannose salvage pathways to
glycosylation in PMI-deficient mouse embryonic fibroblast
cells
Naonobu Fujita
1
, Ayako Tamura
1
, Aya Higashidani
1
, Takashi Tonozuka
1
, Hudson H. Freeze
2
and
Atsushi Nishikawa
1
1 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Japan
2 Tumor Microenvironment Program, Burnham Institute for Medical Research, La Jolla, CA, USA
Eukaryotic cells contain mannose mainly in the form
of N-linked oligosaccharides and glycophospholipid
anchors [1,2]. The only known pathway providing
GDP-mannose for these molecules requires the follow-
ing conversions: mannose 6-phosphate to mannose
1-phosphate to GDP-mannose and dolichyl-P-mannose
[3,4]. In mammals, mannose 6-phosphate can be
formed in several ways. The first, primary, and by far
the best-known, way is via the following conversions:
Glc to Glc6P to Fru6P to mannose 6-phosphate.
Phosphomannose isomerase (PMI) (which catalyzes
the Fru6P to mannose 6-phosphate conversion) has an
important role in this pathway. The second is by direct

a significant contribution to N-glycosylation. When these cells were grown
in mannose-free medium along with the mannosidase inhibitor, swainso-
nine, to block the salvage pathways, N-glycosylation of DNase I was
almost completely eliminated. Adding 13 lm mannose to the medium
completely restored normal glycosylation. Treatment with bafilomycin A
1
,
an inhibitor of lysosomal acidification, also markedly reduced N-glycosyla-
tion of DNase I, but in this case only 8 lm mannose was required to
restore full glycosylation, indicating that a nonlysosomal source of man-
nose made a significant contribution. Glycosylation levels were greatly also
reduced in glycoconjugate-free medium, when endosomal membrane traf-
ficking was blocked by expression of a mutant SKD1. From these data, we
conclude that PMI-null cells can salvage mannose from both endogenous
and external glycoconjugates via lysosomal and nonlysosomal degradation
pathways.
Abbreviations
CDG, congenital disorder of glycosylation; GFP, green fluorescent protein; LLO, lipid-linked oligosaccharide; MOI, multiplicity of infection;
PMI, phosphomannose isomerase.
788 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS
salvaged from glycoconjugates that are degraded
within the same cell. An additional minor pathway
may be conversion of GDP-fucose to GDP-mannose
via unstable intermediate GDP-4-keto-6-deoxy-man-
nose [6]. It has previously been assumed that most of
the mannose in macromolecules is derived from glu-
cose [7]. This assumption was based on the universal
distribution of PMI [8], and the fact that PMI is essen-
tial for yeast growth in the absence of mannose [9].
However, exogenous mannose can contribute signifi-

[19] in PMI-null cells. Wild-type DNase I has two
potential N-linked glycosylation sites. Although the
N-terminal Asn18–Ala–Thr sequon is fully glycosylat-
ed, the C-terminal Asn106–Asp–Ser sequon differs in
tissues and cultured cells [19]. The occupation of the
C-terminal sequon might reflect the glycosylation capa-
bility of cells. Using PMI-null cells, we can examine the
mannose salvage pathways independently of glucose
interconversion at a physiological concentration of
glucose. In this article, we demonstrate that mannose
salvage pathways make significant contributions to
glycosylation, and that the degradation of glycoconju-
gates occurs mainly at low pH in the lysosomes. We
also show that, when cells were incubated in medium
supplemented with 10% fetal bovine serum, the pre-
dominant source of salvaged mannose was degradation
of endogenous glycoconjugates.
Results
Determination of glycosylation efficiency using
the mutant DNase I expression method
Adenovirus for expression of bovine mutant DNase I
that has only one N-linked glycosylation site was
infected into various cultured cells, and the glycosyla-
tion efficiency of expressed DNase I was determined
[19]. As shown in Fig. 1A, the percentage of glycosy-
lated molecules showed an inherent value depending
on the cells. When another mutant DNase I was
expressed in these cells, e.g. with the Asn106–Ser–Thr
sequon instead of the Asn106–Asp–Ser sequon, the
glycosylation efficiency in these cells was almost

has
more than 10 potential N-glycosylation sites. It is
noteworthy that the percentage of glycosylated
DNase I correlated well with that of the immunopre-
cipitated protein. Thus, the glycosylation efficiency of
this mutant DNase I with the Asn106–Asp–Ser se-
quon seemed to reflect the glycosylation capability of
the cells.
Glycosylation efficiency of mutant DNase I
expressed in CDG-Ib fibroblast cells
We measured the glycosylation efficiency of DNase I
in CDG-Ib fibroblast cells, mouse wild-type fibroblast
cells, and PMI-null embryonic fibroblast cells. CDG-Ib
N. Fujita et al. Intracellular mannose salvage pathways
FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 789
cells, owing to a substantial deficiency in PMI activity,
do not have enough LLO, which results in unoccupied
N-linked glycosylation sequons. In the absence of
exogenously supplied mannose, GDP-mannose levels
are markedly lower in CDG-Ib cells [20]. In PMI-null
cells, mannose 6-phosphate cannot be supplied from
the glycolytic pathway via PMI, so when PMI-null
cells are cultured in mannose-free medium, mannose
6-phosphate for glycosylation must be salvaged from
elsewhere. To remove the effects of free mannose in
the medium, cells were cultured in MEM supplemented
with 10% dialyzed fetal bovine serum for 24 h, and
glycosylation analyses were then performed. We pre-
dicted that the glycosylation efficiency would be lower
in CDG-Ib cells and decreased markedly in PMI-null

S]Met and [
35
S]Cys. (A) The protein was immuno-
precipitated from conditioned medium with rabbit anti-DNase I
serum and protein A–agarose beads, and the eluant was subjected
to SDS ⁄ PAGE. In the first panel, lane 1 is intact immunoprecipitat-
ed protein, and lanes 2 and 3 are endo-b-N-acetylglucosamini-
dase-treated and PNGase F-treated samples, respectively. DNase I
having different types of N-glycan is usually detected as a
broad band [19]. The arrow indicates the location of non-glycosylat-
ed DNase I, and ‘]’ shows the migration of the singly glycosylated
DNase I. The percentage of glycosylated molecules is indicated
under the picture. (B) Upper panel: determination of glycosylation
efficiency of mutant DNase I in several fibroblast cells derived from
CDG type I patients. 42F is a human control fibroblast cell line. The
percentage of glycosylated molecules, quantified by densitometry,
is indicated under the picture. Lower panel: SDS ⁄ PAGE of immuno-
precipitated integrin b
1
derived from several CDG type I cells.
35
S-labeled cells obtained from above experiment were lysed
(100 m
M Tris ⁄ HCl, 150 mM NaCl, 1% NP-40 buffer, pH 8.0, con-
taining Roche protease inhibitor cocktail) and immunoprecipitated
using monoclonal antibody to integrin b
1
and protein A–agarose
beads. The precipitate was subjected to SDS ⁄ PAGE using 7%
polyacrylamide gel.

]
Fig. 2. Effect of swainsonine and bafilomycin A
1
on glycosylation.
CDG-Ib fibroblast cells, mouse wild-type embryonic fibroblast cells
and PMI-null fibroblast cells were incubated for 24 h in MEM sup-
plemented with 10% dialyzed fetal bovine serum with or without
10 l
M swainsonine (SW) or 100 nM bafilomycin A
1
(Baf.A1) prior to
metabolic labeling. The percentages of glycosylated molecules
were analyzed as described in Experimental procedures. (A)
SDS ⁄ PAGE analysis representative of three experiments with simi-
lar results. The arrow indicates the location of nonglycosylated
DNase I. (B) The bands in (A) were quantified by densitometry, and
percentages of the glycosylated molecules were calculated. Values
shown are means of five independent experiments.
Intracellular mannose salvage pathways N. Fujita et al.
790 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS
To investigate the relative contribution of lysosomal
a-mannosidases to mannose salvage pathways in PMI-
null cells, they were treated with either swainsonine or
bafilomycin A
1
, and glycosylation analysis was per-
formed. Swainsonine is an indolizidine alkaloid that
acts as a reversible inhibitor of lysosomal a-mannosi-
dase and of the Golgi complex a-mannosidase II [21].
Bafilomycin A

along
with the indicated concentration of mannose for 12 h
prior to metabolic labeling. The percentages of
glycosylated molecules were then measured (Fig. 3). As
expected, the glycosylation efficiencies increased in
swainsonine-treated and bafilomycin A
1
-treated PMI-
null cells with increasing mannose concentrations in
the medium. About 13 lm and 8 lm mannose were
sufficient to fully restore glycosylation in swainsonine-
treated and bafilomycin A
1
-treated PMI-null cells,
respectively. From these results, we conclude that the
reduction in glycosylation efficiency in swainso-
nine-treated and bafilomycin A
1
-treated PMI-null cells
is caused by blockade of the mannose salvage path-
ways and not of the glycoprotein maturation steps.
Overexpression of SKD1
E235Q
strongly inhibited
core glycosylation in PMI-null cells
In mannose salvage pathways, important sources of
mannose are probably glycoconjugates, which are
transported to lysosomes by a membrane trafficking
process. To test the involvement of membrane traffick-
ing in mannose salvage pathways, we examined the

1 0 (µ
M)
PMI-null
WT
Man conc.
SW
A

B
0
20
40
60
80
100
0 5 10 15 20
Mannose conc. (µ
M)
Glycosylation efficiency (%)
PMI-null (SW)
WT (SW)
PMI-null (Baf.A1)
WT (Baf.A1)
Fig. 3. Mannose titration in swainsonine-treated or bafilomycin A
1
-
treated PMI-null cells. PMI-null cells were cultured with 10 l
M
swainsonine or 100 nM bafilomycin A
1

and the glycosylation assay was then performed. As
shown in Fig. 5A, although glycoconjugates were
almost completely removed from the culture medium,
the percentage of glycosylated molecules in PMI-null
cells was only slightly reduced. We obtained the same
result when preincubation in serum-free medium was
extended to 24 h (data not shown). This indicates that,
when the culture medium does not contain fetal bovine
serum, substantial amounts of mannose can be derived
from endogenous rather than exogenous glycoconju-
gates.
If endogenous glycoconjugates are the main source
of mannose, the percentage of glycosylated molecules
should be reduced with time when PMI-null cells are
cultured in mannose-free medium,. Prior to metabolic
labeling, PMI-null cells were incubated with MEM
supplemented with 10% dialyzed fetal bovine serum
for 0, 12, 24, 36 or 48 h (Fig. 5B), and glycosylation
assays were then performed. As shown in Fig. 5C,
increasing the preincubation time reduced the percentage
0
20
40
60
80
100
120
W
T PMI n
u

, cells
were coinfected with AxCALSKD
EQ
at an
MOI of 0, 1000, or 2000, and AxCANCre at
an MOI of 150. (A) Twenty-four hours post-
infection, the cells were washed twice with
NaCl ⁄ P
i
and fixed with 4% paraformalde-
hyde for 15 min. Fluorescent images were
obtained with a Zeiss Axiocam controlled by
AXIOVISON software. (B) Prior to metabolic
labeling, the cells were incubated with
MEM supplemented with 10% dialyzed
fetal bovine serum, containing AxCALSKD
EQ
and AxCANCre at the indicated MOI. The
glycosylation analysis was performed as
described in Experimental procedures. The
results are presented as percentage of
untreated cells, which was defined as
100%.
Intracellular mannose salvage pathways N. Fujita et al.
792 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS
MEM+ 20% FBS
MEM + 10% dialyzed FBS
012243648
60
Pre-incubation time (h)

0 h
24 h
48 h
G0 1 2 3
Glycoprotein Total protein
G. R. 1 0.94 0.64 (Gl
y
co
p
rotein/Total
p
rotein)
F. I. 100 87 56 % 100 93 87 %
M0
24 48 h
M0
24 48 h
82 k
42 k
180 k
Fig. 5. The contribution of serum glycoproteins in culture medium to N-glycosylation. (A) To remove glycoproteins from the culture medium,
10% dialyzed fetal bovine serum was replaced with 1% BSA or 2% TCH. PMI-null cells were incubated for 12 h in MEM supplemented with
1% BSA or 2% TCH prior to metabolic labeling. The percentages of glycosylated molecules were analyzed as described in Experimental proce-
dures. The number of glycosylated molecules is presented as percentage of the value in 10% dialyzed fetal bovine serum. (B) Scheme of the
time course for an experiment. After 0, 12, 24, 36 or 48 h of incubation with MEM supplemented with 10% dialyzed fetal bovine serum (indi-
cated with dotted line), the glycosylation analyses were performed. (C) Time course of the reduction in glycosylation efficiency of DNase I in
PMI-null cells. The medium was replaced every 4 h with fresh MEM supplemented with 10% dialyzed fetal bovine serum. Values represent
mean ± SD of three independent experiments. (D) LLO patterns of cells harvested after 0, 24 and 48 h of incubation with MEM supplemented
with 10% dialyzed fetal bovine serum. The cells were collected, and LLOs were labeled with 2-aminopyridine and analyzed using HPLC accord-
ing to the method described in Experimental procedures. Ten per cent volume of the product of each sample was injected into the column. The

showed that the percentage of glycosylated protein in
cells decreased to 64% and the total amount of glyco-
protein dropped to nearly half the initial amount. On
the other hand, the amount of glycoprotein in the
medium did not differ substantially from the beginning
to the end of the experiment, because the medium was
changed every 4 h. From these findings, we conclude
that PMI-null cells incubated in medium supplemented
with 10% fetal bovine serum can salvage mannose
from endogenous glycoconjugates, but that the amount
is insufficient to maintain normal protein synthesis
(growth) or normal levels of LLO.
Discussion
In this study, the mannose salvage pathways have been
systematically investigated in PMI-null cells. The four
main findings are as follows: (a) the contribution of
mannose salvage pathways to glycosylation is quite
substantial; (b) glycoconjugate degradation mainly
occurs in lysosomes under low-pH conditions; (c) gly-
coconjugates are transported to lysosomes via endo-
somal trafficking pathways; and (d) when cells are
incubated in medium supplemented with 10% fetal
bovine serum, mannose can be derived from endoge-
nous glycoconjugates.
The unexpected finding that normal and CDG-Ib
cells showed nearly equal levels of glycosylation
(Fig. 2) led us to investigate the mannose salvage path-
way in mammalian cells. PMI-null cells cannot gener-
ate mannose from glucose, and they are forced to rely
on mannose salvage pathways. This allowed us exam-

protein synthesis (Fig. 6). The mannose titration
results and those of a previous report [10] indicate that
a low concentration of mannose is sufficient for proper
glycosylation in fibroblast cells. Furthermore, we also
observed that a substantial amount of mannose can be
Glucose
Mannose
GDP-
-6-P
-6-P
-1-P
Fru-6-P
Lysosome
PMI
PMI
Glycolysis/
TCA cycle
LLO synthesis/
Glycosylation/
Processing
Glycoprotein
degradation
ER
Glucose transporter Mannose transporter
M
M
M
M
M
G

therefore, further research is required to clarify these
points.
Although this study was focused on the lysosomal
mannose salvage pathway, which is dependent on
membrane trafficking, there are also two membrane
traffic-independent mannose salvage pathways [30]. The
first involves the free mannose that is generated during
glycoprotein maturation steps. As mammalian cells
generally contain higher proportions of complex oligo-
saccharides than unprocessed high-mannose-type
chains, it is clear that most of the nine mannose residues
initially incorporated into the LLO precursor will be lost
as free mannose. The second involves the free oligo-
saccharides that are generated in the cytosol, and are
products of LLO breakdown, glycopeptides and incor-
rectly folded glycoproteins generated during quality
control screening of the biosynthesis of glycoproteins in
the endoplasmic reticulum [31]. Indeed, the glycosyla-
tion efficiency in PMI-null cells was reduced to almost
zero both by swainsonine and bafilomycin A
1
treatment,
but there were considerable differences in the mannose
titration curves (Fig. 3B). This distinction may be due
to differences in the mechanism of action of swainsonine
and bafilomycin A
1
. Swainsonine inhibits both lyso-
somal and nonlysosomal mannosidases, whereas bafilo-
mycin A

were from Waters (Milford, MA, USA) and Supelco (Belle-
fonte, PA, USA) respectively, and fetal bovine serum was
obtained from ICN Biomedicals (Costa Mesa, CA, USA).
Other media and reagents were from Sigma (St Louis, MO,
USA).
Plasmid and adenovirus preparation
Previously, we described an N-glycosylation efficiency assay
[19]. In this work, we used 1-delta wild-type bovine
DNase I, in which Asn18 was exchanged for Glu, with only
one glycosylation site, Asn106–Asp–Ser. To obtain higher
reproducibility, an adenovirus expression system of mutant
DNase I was constructed as follows. Adenoviruses bearing
mutant bovine DNase I was prepared using the ViraPower
Adenovirus Expression System according to the manufac-
turer’s instructions. Briefly, the mutant bovine DNase I
cDNAs were subcloned into the pENTER 1A vector. After
purification of the plasmids, the cDNA inserts were trans-
ferred to the pAd ⁄ CMV ⁄ V5-DEST vector by means of the
Gateway system using LR clonase. The plasmids were puri-
fied and digested with the PacI restriction enzyme. One
microgram of linearized plasmid was diluted with 200 lL
of Opti-MEM, and then mixed with 200 lL of lipofecta-
mine solution, dissolving 4 lL of Lipofectamine 2000 in
Opti-MEM, and transfected into subconfluent 293A cells in
1 mL of Opti-MEM in six-well plates. The 293A cells were
then cultured for 8 days in DMEM supplemented with
10% fetal bovine serum. The medium was replaced every
2 days. When most cells became detached from the plates,
the cells and culture medium were harvested together,
freeze–thawed three times, and centrifuged (1500 g for

N. Fujita et al. Intracellular mannose salvage pathways
FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 795
adenoviruses was replaced with 1 mL of MEM supple-
mented with 10% dialyzed fetal bovine serum. Following
an additional incubation for 12 h, the medium was again
replaced with 1 mL of MEM supplemented with 10% dia-
lyzed fetal bovine serum. After incubation for another
12 h, the cells were washed twice with NaCl ⁄ P
i
and incu-
bated for 4 h at 37 °C with 0.7 mL of MEM without
Met ⁄ Cys but supplemented with 10% dialyzed fetal bovine
serum and 2.65 MBq of [
35
S]Met ⁄ Cys labeling mixture.
The culture medium was then harvested.
Immunoprecipitation and glycosylation analysis
The extent of glycosylation of mutant DNase I was mea-
sured as previously described [19]. Briefly, the harvested cell
culture medium was incubated with 1 lL of anti-DNase I
serum overnight. Then, 20 lL of a 50% protein A–agarose
bead suspension was added. After 1 h of additional rota-
tion, the beads were washed three times. The immunopre-
cipitated protein was then eluted from the beads in 2X
SDS ⁄ PAGE sample buffer by boiling for 5 min. The eluted
samples were separated by SDS ⁄ PAGE using 12% poly-
acrylamide gel, and the intensities of the bands correspond-
ing to DNase I were quantified using a BAS1000 bioimage
analyzer (Fuji Film Co., Tokyo, Japan).
Glycosidase digestion

concentrations of mannose (0–20 lm) for 12 h. Metabolic
labeling was also performed in the presence of 10 lm
swainsonine or 100 nm bafilomycin A
1
and the indicated
concentrations of mannose. The glycosylation analysis was
then performed as described above.
GFP–SKD1
E235Q
overexpression
A Cre ⁄ loxP inducible system was utilized to express
SKD1
E235Q
[26], because constitutive expression of
SKD1
E235Q
is toxic for 293A cells, in which recombinant
adenoviruses are grown. Twenty-four hours after infection
with adenoviruses bearing mutant DNase I at an MOI of
300, cells were washed and coinfected with adenoviruses
bearing SKD1
E235Q
(AxCALSKD
EQ
) at an MOI of 0, 500,
1000, or 2000, and Cre recombinase (AxCANCre) at an
MOI of 150. After incubation for 12 h, the medium con-
taining adenoviruses was replaced with 1 mL of MEM
containing 10% dialyzed fetal bovine serum. Following an
additional incubation for 12 h, the glycosylation analysis

ation efficiency of DNase I and total cell protein were
analyzed. In this experiment, medium was replaced every
4 h with fresh MEM supplemented with 10% dialyzed fetal
bovine serum. For measurement of glycosylation of total
cell protein, collected cells were lysed by 8 m urea contain-
ing 2% Chaps, and then each lysate was subjected to
SDS ⁄ PAGE using 12% polyacrylamide gel. First, glycopro-
teins were stained with Pro-Q Emerald 300, and second,
Intracellular mannose salvage pathways N. Fujita et al.
796 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS
total proteins were stained with the SYPRO Ruby protein
gel stain kit according to the manufacturer’s instructions.
Using a Lumino LAS-3000 imaging analyzer and multi
gauge v2.1 software (Fuji Film), the stained proteins were
imaged.
Analysis of LLOs
Approximately 1.0 · 10
7
harvested cells were suspended
in methanol and dried under N
2
. Afterwards, LLOs
were extracted as previously described [32]. Briefly, LLOs
were extracted in chloroform ⁄ methanol ⁄ water (CMW;
10 : 10 : 3), and the materials in the CMW extract were
treated with weak acid to generate soluble oligosaccha-
rides. The hydrolysates were then loaded onto a C
18
SepPak column directly connected to a 3 mL ENVI-CARB
solid-phase extraction tube to remove residual salt and lip-

at the same flow rate. Elution was monitored by fluores-
cence (excitation wavelength, 320 nm; emission wavelength,
400 nm). Each peak was identified by comparison with a
standard pyridylaminated oligosaccharide elution time.
Acknowledgements
We express our gratitude to Dr Tamotsu Yoshimori
for providing adenoviruses bearing Cre-recombinase
and GFP–SKD1
E235
, to Dr Sumihiro Hase for pro-
viding standard pyridylaminated oligosaccharides, and
to Mr Kazuyuki Iimura for technical assistance. To
Dr Nobuhiro Takahashi for permission to use his
laboratory for fluorescence microscopy. This work
was supported by CREST of JST, the National Insti-
tutes of Health grant R01 DK55695, and the Rocket
Williams Fund (HHF).
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